Specific interactions between proteins play pivotal roles in virtually all biological processes, yet our understanding of these important molecular interactions is still rather limited. Until recently, the only technique capable of yielding atomic-level information about biological macromolecules was x-ray diffraction. The application of two-dimensional nuclear magnetic resonance (2DNMR) spectroscopy to the study of small-to- medium-sized proteins in solution offers a second method for structural studies. This proposal involves the use of 2DNMR spectroscopy in both detailed structure/function studies and in the study of specific protein-protein interactions. Two different systems will be studied: 1) the phosphoenolpyruvate-dependent sugar transport system of bacteria, with particular focus on the phosphocarrier protein, histidine-containing protein (HPr) and 2) the Ca2+-regulatory protein, calmodulin, and its interactions with target enzymes. The proposed structure/function studies of HPr involve a combined approach of site-directed mutagenesis and 2DNMR. The complete sequence-specific assignments for the HPr spectrum (determined in the original granting period) will serve as the basis of these studies. This approach promises to yield much information relating the functional and structural consequences of single amino acid changes to a protein. New applications of 2DNMR experiments are proposed to study the interactions between HPr and its phosphoryl acceptor protein, factor III. 2DNMR studies to calmodulin-target enzyme interactions are made possible by the recent discovery of short linear sequences from target enzymes such as myosin light chain kinase that possess the properties expected for calmodulin-binding domains. These studies will take advantage of solid-phase peptide synthesis technology, allowing for "mutation" of the sequences as well as incorporation of specific isotopes (i.e., 2H, 13C, 15N) to be used with special "spectral editing" techniques.